Fig. 4: Effects of CD39 blocking or knockout on ATP metabolism and on the immunosuppressive effects of HTLV-1-infected cell lines.

a, b Each cell line was cultured with 1 mM ATP, with or without the CD39 inhibitors ARL (left panel) or POM1 (right panel), for 1 h at the indicated concentrations. All cell lines were CD39 positive. Results are expressed as the mean ± SD of at least three independent experiments. c The CD39-positive cell line MJ was introduced with empty vector (mock) or CRISPR/CAS9 knockout plasmid including a GFP expression site. Flow cytometry confirmed CD39 knockout in the GFP-positive fraction of CD39KO transfected cells, and CD39 expression in mock transfected cells. The GFP-positive cells were selected using a FACS cell sorter and were used in the following experiments. d As in Fig. 3, ATP consumption was measured in mock or CD39KO transfected MJ cells. Bar heights indicate the mean ± SD of the % ratio of remaining ATP relative to supplemented amount, from triplicate experiments. e Suppressive activity of mock/CD39KO MJ cells. CellTrace^TM Violet-labeled CD25⁻CD4⁺ (left panels) or CD8⁺ T cells (1 × 10⁴) were stimulated with soluble anti-CD3 and anti-CD28 mAbs in the presence of irradiated PBMCs excluding CD3 positive cells. Mock or CD39KO MJ cells (1 × 10⁴) were added to the cultures. After 3 days of culture, the violet intensity was measured by FACS as an indicator of cell division. f Bar charts show the frequency (%) of responder T cells, which have undergone cell division, with or without MJ cells or with each transfected MJ cell line. The results are expressed as the mean ± SD of triplicate experiments. For a, b, and f, data were compared using Student’s t test; ns not statistically significant; *P < 0.05; **P < 0.01; and ***P < 0.001.